Nanoparticles that fluoresce when excited by an appropriate energy source, such as a laser are powerful detection tools in a variety of life science applications ranging from Western blot detection of proteins, visualization of cell migration, flow cytometric analysis to in vivo imaging. Such nanoparticles also form foundational components in LEDs, solar cells, transistors, and diode lasers. However, the fluorescent nanoparticles currently available suffer from intermittent fluorescence or a stochastic blinking on and off of their fluorescence when the nanoparticles are excited. This blinking on and off limits the robustness of the signal as both the timing and duration of the on/off periods are unpredictable. For single particle or single molecule analysis, the blinking properties limit the usefulness of the fluorescent nanoparticles. Similarly, blinking results in significant hurdles (that can be insurmountable) in ultra high-throughput applications using a population of nanoparticles, due to unpredictable variations in signal intensity resulting from the nanoparticles intermittently toggling between an on/off state.
While not being bound to a particular theory, one premise attributes the blinking behavior to the steep electronic interface between the particle and the “outside world.” Blinking therefore may result from the temporary loss of a photo-electron or Auger electron (or a hole) from the particle core to the surrounding matrix, for example by Auger ejection or charge tunneling, or to electron capture by surface-related traps, producing a charged state. When the nanoparticle is in a charged state, emission is turned off. Once charge neutrality is restored, emission turns on, resulting in the characteristic blinking. A key observation driving these theories is that blinking worsens as a function of excitation power. In other words, as the excitation power increases, the blinking typically increases as well. Such observations suggest that blinking occurs with either simultaneous or sequential excitation by two or more photons per excited state. Thus, one useful approach to suppress blinking can be to prevent electrons from escaping the particle core during or following a multi-photon event.
There is a need to develop approaches to provide small (such that they are useful in fluorescence resonance energy transfer applications) and stable nanoparticles which address the problems posed by nanoparticle fluorescent intermittency (as this intermittency complicates the reliable use of “blinking” nanoparticles as a single photon light source for quantum informatics and as biolabels for real-time monitoring of single biomolecules).